Comparative evaluation of two lures to attract female mosquitoes in an urban natural reserve during daytime.

The most widely used attractant to capture adult female mosquitoes is CO2. However, there are also baits available on the market that emit a scent resembling human skin. These baits were specifically designed to attract highly anthropophilic species such as Aedes albopictus and Aedes aegypti. In this study, we compare the effectiveness of CDC traps baited either with CO2 or with a commercial blend simulating skin odor, BG-Sweetscent, for trapping female mosquitoes during daylight hours in an urban reserve in the City of Buenos Aires. We employed a hurdle generalized linear mixed model to analyze trap capture probability and the number of mosquitoes captured per hour, considering the effects of attractant, mosquito species, and their interaction. Traps baited with CO2 captured ten mosquito species, while those baited with BG-Sweetscent captured six in overall significantly lower abundance. The odds of capturing mosquitoes were 292% higher for the CO2-baited traps than for those baited with BG-Sweetscent. No evidence of a combined effect of attractant type and species on female mosquito captures per hour was found. Results indicated that CDC traps baited with CO2 were more effective than those baited with BG-Sweetscent in capturing more mosquito species and a higher number of mosquitoes within each species, even if the species captured with CO2 exhibited a certain level of anthropophilia. This result has practical implications for mosquito surveillance and control in urban natural reserves.

Antiviral Activities of Heparan Sulfate Mimetic RAFT Polymers Against Mosquito-borne Viruses.

Mosquito-borne viruses are a major worldwide health problem associated with high morbidity and mortality rates and significant impacts on national healthcare budgets. The development of antiviral drugs for both the treatment and prophylaxis of these diseases is thus of considerable importance. To address the need for therapeutics with antiviral activity, a library of heparan sulfate mimetic polymers was screened against dengue virus (DENV), Yellow fever virus (YFV), Zika virus (ZIKV), and Ross River virus (RRV). The polymers were prepared by RAFT polymerization of various acidic monomers with a target MW of 20 kDa (average Mn ∼ 27 kDa by GPC). Among the polymers, poly(SS), a homopolymer of sodium styrenesulfonate, was identified as a broad spectrum antiviral with activity against all the tested viruses and particularly potent inhibition of YFV (IC50 = 310 pM). Our results further uncovered that poly(SS) exhibited a robust inhibition of ZIKV infection in both mosquito and human cell lines, which points out the potential functions of poly(SS) in preventing mosquito-borne viruses associated diseases by blocking viral transmission in their mosquito vectors and mitigating viral infection in patients.

Effects of chironomid larvae density and mosquito biocide on methane and carbon dioxide dynamics in freshwater sediments.

Small lentic water bodies are important emitters of methane (CH4) and carbon dioxide (CO2), but the processes regulating their dynamics and susceptibility to human-induced stressors are not fully understood. Bioturbation by chironomid larvae has been proposed as a potentially important factor controlling the dynamics of both gases in aquatic sediments. Chironomid abundance can be affected by the application of biocides for mosquito control, such as Bti (Bacillus thuringiensis var. israelensis). Previous research has attributed increases in CH4 and CO2 emissions after Bti application to reduced bioturbation by chironomids. In this study, we separately tested the effect of chironomid bioturbation and Bti addition on CH4 production and emission from natural sediments. In a set of 15 microcosms, we compared CH4 and CO2 emission and production rates with high and low densities of chironomid larvae at the bioturbating stage, and standard and five times (5x) standard Bti dose, with control sediments that contained neither chironomid larvae nor Bti. Regardless of larvae density, chironomid larvae did not affect CH4 nor CO2 emission and production of the sediment, although both rates were more variable in the treatments with organisms. 5xBti dosage, however, led to a more than three-fold increase in CH4 and CO2 production rates, likely stimulated by bioavailable dissolved carbon in the Bti excipient and priming effects. Our results suggest weak effects of bioturbating chironomid larvae on the CH4 and CO2 dynamics in aquatic ecosystems. Furthermore, our results point out towards potential functional implications of Bti for carbon cycling beyond those mediated by changes in the macroinvertebrate community.

A perspective on the impacts of microplastics on mosquito biology and their vectorial capacity.

Microplastics (plastic particles <5 mm) permeate aquatic and terrestrial ecosystems and constitute a hazard to animal life. Although much research has been conducted on the effects of microplastics on marine and benthic organisms, less consideration has been given to insects, especially those adapted to urban environments. Here, we provide a perspective on the potential consequences of exposure to microplastics within typical larval habitat on mosquito biology. Mosquitoes represent an ideal organism in which to explore the biological effects of microplastics on terrestrial insects, not least because of their importance as an infectious disease vector. Drawing on evidence from other organisms and knowledge of the mosquito life cycle, we summarise some of the more plausible impacts of microplastics including physiological, ecotoxicological and immunological responses. We conclude that although there remains little experimental evidence demonstrating any adverse effect on mosquito biology or pathogen transmission, significant knowledge gaps remain, and there is now a need to quantify the effects that microplastic pollution could have on such an important disease vector.

Measuring insecticide resistance in a vacuum: exploring next steps to link resistance data with mosquito control efficacy.

Insecticide resistance is a great challenge facing mosquito operational control agencies across the United States, where few active ingredients with unique modes of action are available for use, increasing resistance pressure and further hampering resistance management strategies. Emergence and expansion of insecticide resistance in mosquitoes can be detected by resistance monitoring programs; however, there are gaps in our knowledge regarding the link between resistance bioassay results and operational control outcomes. Here, we review both public health and agricultural studies on pesticide resistance bioassays and control outcomes. A discussion on the main gaps in our knowledge of insecticide resistance and a review of resistance management practices is also presented. We conclude with research questions that can advance our understanding of resistance monitoring and control.

Standardised bioassays reveal that mosquitoes learn to avoid compounds used in chemical vector control after a single sub-lethal exposure.

Vector-borne diseases are worldwide public health issues. Despite research focused on vectorial capacity determinants in pathogen transmitting mosquitoes, their behavioural plasticity remains poorly understood. Memory and associative learning have been linked to behavioural changes in several insect species, but their relevance in behavioural responses to pesticide vector control has been largely overlooked. In this study, female Aedes aegypti and Culex quinquefasciastus were exposed to sub-lethal doses of 5 pesticide compounds using modified World Health Organization (WHO) tube bioassays. Conditioned females, subsequently exposed to the same pesticides in WHO tunnel assays, exhibited behavioural avoidance by forgoing blood-feeding to ensure survival. Standardized resting site choice tests showed that pre-exposed females avoided the pesticides smell and choose to rest in a pesticide-free compartment. These results showed that, following a single exposure, mosquitoes can associate the olfactory stimulus of pesticides with their detrimental effects and subsequently avoid pesticide contact. Findings highlight the importance of mosquito cognition as determinants of pesticide resistance in mosquito populations targeted by chemical control.

Mosquito population structure, pathogen surveillance and insecticide resistance monitoring in urban regions of Crete, Greece.

BACKGROUND: In Greece vector borne diseases (VBD) and foremost West Nile virus (WNV) pose an important threat to public health and the tourist industry, the primary sector of contribution to the national economy. The island of Crete, is one of Greece's major tourist destinations receiving annually over 5 million tourists making regional VBD control both a public health and economic priority. METHODOLOGY: Under the auspices of the Region of Crete, a systematic integrative surveillance network targeting mosquitoes and associated pathogens was established in Crete for the years 2018-2020. Using conventional and molecular diagnostic tools we investigated the mosquito species composition and population dynamics, pathogen infection occurrences in vector populations and in sentinel chickens, and the insecticide resistance status of the major vector species. PRINCIPAL FINDINGS: Important disease vectors were recorded across the island including Culex pipiens, Aedes albopictus, and Anopheles superpictus. Over 75% of the sampled specimens were collected in the western prefectures potentially attributed to the local precipitation patterns, with Cx. pipiens being the most dominant species. Although no pathogens (flaviviruses) were detected in the analysed mosquito specimens, chicken blood serum analyses recorded a 1.7% WNV antibody detection rate in the 2018 samples. Notably detection of the first WNV positive chicken preceded human WNV occurrence in the same region by approximately two weeks. The chitin synthase mutation I1043F (associated with high diflubenzuron resistance) was recorded at an 8% allelic frequency in Lasithi prefecture Cx. pipiens mosquitoes (sampled in 2020) for the first time in Greece. Markedly, Cx. pipiens populations in all four prefectures were found harboring the kdr mutations L1014F/C/S (associated with pyrethroid resistance) at a close to fixation rate, with mutation L1014C being the most commonly found allele (≥74% representation). Voltage gated sodium channel analyses in Ae. albopictus revealed the presence of the kdr mutations F1534C and I1532T (associated with putative mild pyrethroid resistance phenotypes) yet absence of V1016G. Allele F1534C was recorded in all prefectures (at an allelic frequency range of 25-46.6%) while I1532T was detected in populations from Chania, Rethymnon and Heraklion (at frequencies below 7.1%). Finally, no kdr mutations were detected in the Anopheles specimens included in the analyses. CONCLUSIONS/SIGNIFICANCE: The findings of our study are of major concern for VBD control in Crete, highlighting (i) the necessity for establishing seasonal integrated entomological/pathogen surveillance programs, supporting the design of targeted vector control responses and; ii) the need for establishing appropriate insecticide resistance management programs ensuring the efficacy and sustainable use of DFB and pyrethroid based products in vector control.

Cananga odorata (Magnoliales: Annonaceae) Essential Oil Produces Significant Avoidance Behavior in Mosquitoes.

Essential oil of Cananga odorata Hook. F. & Tomson is a source of insect repellent, but contact irritancy and noncontact repellency actions that stimulate insect's avoidance behavior (escape away from chemical source after direct physical contact or without making physical contact, respectively) have not been investigated. Therefore, an excito-repellency test chamber was used for measuring avoidance behavior of four insectary-reared mosquito species (Diptera: Culicidae) that escape from esposure to four concentrations (0.5, 1.0, 2.5, and 5.0% v/v) of C. odorata oil. The oil strongly repelled both Culex quinquefasciatus Say (85-97% escape) and Anopheles minimus Theobald (97-99%) at high concentrations (2.5-5.0%). For Anopheles dirus Peyton & Harrison and Aedes aegypti (L.), highest repellency (64 and 39% escape, respectively) was demonstrated at 2.5% concentration. For contact irritancy, the oil produced relatively high percent escape found in Cx. quinquefasciatus (90-100% escape) and An. minimus (83-100%). Whereas moderate contact irritancy was observed against An. dirus (40-50% escape) and Ae. aegypti (51-59%). The percent escape was then adjusted with repellency to estimate the effect of contact irritancy alone. We found that highest contact irritancy was presented at 0.5% concentration against An. minimus (67% escape). Knockdown and toxic actions were only found in Anopheles mosquitoes at 5.0% concentration. The results revealed that An. minimus and Cx. quinquefasciatus were more prone to be repelled by C. odorata oil. Detailed analysis of oil identified primary compounds as methyl benzoate (14.6%), α-gurjunene (12.8%), p-methyl-anisole (11.3%), and benzyl acetate (9.9%). Further investigations are needed to assess excito-repellency actions of these compounds alone or in combination.

Fish-Released Kairomones Affect Mosquito Oviposition and Larval Life History.

Several species of mosquitoes respond to the presence of kairomones released by larvivorous predators during oviposition habitat selection and larval development. These responses may differ among mosquito species and do not always correlate with larval survival. This study examined the responses of the mosquito Culiseta longiareolata Macquart (Diptera: Culicidae) to kairomones released by three species of fish, Gambusia affinis (Cyprinodontiformes: Poeciliidae), Aphanius mento (Cyprinodontiformes: Cyprinodontidae) and Garra rufa (Cypriniformes: Cyprinidae) during oviposition. In addition, the study examined the effects of kairomones released by Gm. affinis on larval development and survival. Results show that ovipositing female avoided cues from the two larvivorous fish species but not the algivorous Gr. rufa. In addition, developing larvae metamorphosed slower and showed increased mortality when exposed to fish-released kairomones. Culiseta longiareolata larvae are known as dominant competitors, and the straightforward responses of both larvae and adult female to fish-released kairomones may be explained by the lack of additional sources of larval stress other than the presence of predators.

Chemical Composition, Insecticidal and Mosquito Larvicidal Activities of Allspice (Pimenta dioica) Essential Oil.

Essential oils are biologically and environmentally safe pesticidal compounds yielded from aromatic plants. Spices are important sources of essential oils, and they are widely used in the medicine, food, and various other industries. Among the different spices, Allspice (Pimenta dioica) is underexplored in terms of its biological efficacy and a limited number of studies are available on the chemical composition of Allspice essential oil (AEO); thus, the present study evaluated the larvicidal property, the repellency, and the fumigant toxicity against common pests of stored products of AEO. AEO was found to inhibit the survival of larvae of such vectors as Aedis, Culex, and Armigeres species. Further, AEO was found to exert repellant effects against the pests of such stored products as Sitophilus, Callosobruchus, and Tribolium. Similarly, the fumigant toxicity was found to be high for AEO against these species. The contact toxicity of AEO was high against Sitophilus and Callosobruchus. Apart from that, the essential oil was found to be safe against a non-target organism (guppy fishes) and was found to be non-genotoxic in an Allium cepa model. Overall, the results of the present study indicate that the essential oil from Allspice could be used as an environmentally safe larvicidal and biopesticidal compound.

Properties of Mosquito Repellent-Plasticized Poly(lactic acid) Strands.

Poly(lactic acid) (PLA) is an attractive candidate for replacing petrochemical polymers because it is fully biodegradable. This study investigated the potential of PLA as a sustainable and environmentally friendly alternative material that can be developed into commercially viable wearable mosquito repellent devices with desirable characteristics. PLA strands containing DEET and IR3535 were prepared by twin screw extrusion compounding and simultaneously functioned as plasticizers for the polymer. The plasticizing effect was investigated by thermal and rheological studies. DSC studies showed that the addition of DEET and IR3535 into PLA strands reduced the glass transition temperature consistent with predictions of the Fox equation, thus proving their efficiency as plasticizers. The rheology of molten samples of neat PLA and PLA/repellents blends, evaluated at 200 °C, was consistent with shear-thinning pseudoplastic behaviour. Raman studies revealed a nonlinear concentration gradient for DEET in the PLA strand, indicating non-Fickian Type II transport controlling the desorption process. Release data obtained at 50 °C showed initial rapid release followed by a slower, near constant rate at longer times. The release rate data were fitted to a novel modification of the Peppas-Sahlin desorption model.

Reducing mosquito-borne disease transmission to humans: A systematic review of cluster randomised controlled studies that assess interventions other than non-targeted insecticide.

BACKGROUND: Mosquito control interventions are widely used to reduce mosquito-borne diseases. It is unclear what combination of interventions are most effective in reducing human disease. A novel intervention study for Buruli ulcer targeting mosquito vectors was proposed for a Buruli ulcer-endemic area of Victoria, Australia. The local community expressed a preference for avoiding widespread residual spraying of pyrethroids. To inform the design of a future cluster randomised control study (cRCT) for Buruli ulcer prevention in Victoria, we conducted a systematic literature review. AIMS: The aim was to describe cRCT designs which investigated interventions other than non-targeted insecticide for reducing mosquito-borne disease transmission, and comment on the strengths and weaknesses of these study designs. METHODS: Five medical research databases were searched for eligible literature from the earliest available sources up to 5 July 2019 (Medline, Embase, Web of Science, EBM Reviews, CAB Direct). Reference lists of identified studies were hand searched. Eligible studies were cRCTs using targeted chemical or biological mosquito control interventions, or mosquito breeding source reduction, with the occurrence of mosquito-borne disease as an outcome. RESULTS: Eight eligible cRCTs, conducted between 1994-2013 were identified in a variety of settings in the Americas and Asia. Interventions to reduce dengue transmission were mass adult trapping and source reduction. Interventions to reduce malaria transmission were largescale larvicide administration and (topical and spatial) repellent use. Three studies showed the intervention was associated with statistically significant reductions in the disease of interest and entomological indicators. High community engagement with the intervention were common to all three. In two studies, large buffer zones reduced contamination between study arms. Heterogeneity was reduced through increasing study cluster numbers, cluster matching and randomisation. CONCLUSION: High community engagement is vital for a cRCT reducing mosquito-borne disease with a mosquito control intervention. These findings support a mosquito breeding source reduction intervention for Aedes control in a future study of Buruli ulcer prevention if local communities are supportive and very engaged. Regular administration of larvicide to sites unsuited to source reduction may supplement the intervention.

Heterodissemination: precision insecticide delivery to mosquito larval habitats by cohabiting vertebrates.

Conventional larvicide delivery strategies originally developed for permanent and floodwater mosquitoes have proved suboptimal in the small, scattered, and cryptic larval habitats preferred by container-inhabiting Aedes mosquitoes. New methods such as autodissemination, wherein adult mosquitoes spread insecticides to their own larval habitats, have been under study. Another novel delivery method termed heterodissemination, i.e. larvicide delivery by other species sharing the same habitats, has also been proposed. We conducted a proof-of-concept study with four independent experiments using American bullfrogs (Lithobates catesbeianus) and green frogs Lithobates clamitans as carriers of pyriproxyfen, an insect growth regulator, under semi-field conditions in three different locations, two in New Jersey, and one in Utah. Frogs with attached slow-release pyriproxyfen tablets were introduced into outdoor enclosures with water containers. Water samples from the containers were periodically tested using larval Aedes albopictus and Culex pipiens mosquitoes to assess mortality and percent eclosure inhibition. Overall pupal mortality [95% credible intervals] estimated by Bayesian analysis for the treatment group was 73.4% [71.3-75.2] compared to 4.1% [2.9-5.5] for the control group. Mortality within treatment groups in four different experiments ranged from 41 to 100%, whereas control mortalities ranged from 0.5% to 11%. We conclude that heterodissemination is a promising and effective approach deserving of further study.

Effects of Kathon, a Chemical Used Widely as a Microbicide, on the Survival of Two Species of Mosquitoes.

In recent decades, demands for novel insecticides against mosquitoes are soaring, yet candidate chemicals with desirable properties are limited. Kathon is a broad-spectrum isothiazolinone microbicide, but other applications remain uncharacterized. First, we treated larvae of Culex quinquefasciatus and Aedes albopictus, two major mosquito vectors of human viral diseases, with Kathon at 15 mg/L (a concentration considered safe in cosmetic and body care products), and at lower concentrations, and found that Kathon treatment resulted in high mortality of larvae. Second, sublethal concentration of Kathon can cause significantly prolonged larval development of C. quinquefasciatus. Third, we explored the effects of two constituents of Kathon, chloromethylisothiazolinone (CMIT) and methylisothiazolinone (MIT), on the survival of larvae, and found that CMIT was the major toxic component. Further, we explored the mechanisms of action of Kathon against insect cells and found that Kathon reduces cell viability and adenosine triphosphate production but promotes the release of lactate dehydrogenase in Drosophila melanogaster S2 cells. Our results indicate that Kathon is highly toxic to mosquito larvae, and we highlight its potential in the development of new larvicides for mosquito control.

Long-Lasting Insecticidal Nets Incorporating Piperonyl Butoxide Reduce the Risk of Malaria in Children in Western Kenya: A Cluster Randomized Controlled Trial.

Malaria vectors have acquired an enzyme that metabolizes pyrethroids. To tackle this problem, we evaluated long-lasting insecticidal nets incorporating piperonyl butoxide (PBO-LLINs) with a community-based cluster randomized control trial in western Kenya. The primary endpoints were anopheline density and Plasmodium falciparum polymerase chain reaction (PCR)-positive prevalence (PCRpfPR) of children aged 7 months to 10 years. Four clusters were randomly selected for each of the treatment and control arms (eight clusters in total) from 12 clusters, and PBO-LLINs and standard LLINs were distributed in February 2011 to 982 and 1,028 houses for treatment and control arms, respectively. Entomological surveys targeted 20 houses in each cluster, and epidemiological surveys targeted 150 children. Cluster-level permutation tests evaluated the effectiveness using the fitted values from individual level regression models adjusted for baseline. Bootstrapping estimated 95% confidence intervals (CIs). The medians of anophelines per house were 1.4 (interquartile range [IQR]: 2.3) and 3.4 (IQR: 3.7) in the intervention and control arms after 3 months, and 0.4 (IQR: 0.2) and 1.6 (IQR: 0.5) after 10 months, respectively. The differences were -2.5 (95% CI: -6.4 to -0.6) and -1.3 (95% CI: -2.0 to -0.7), respectively. The datasets of 861 and 775 children were analyzed in two epidemiological surveys. The median PCRpfPRs were 25% (IQR: 11%) in the intervention arm and 52% (IQR: 11%) in the control arm after 5 months and 33% (IQR: 11%) and 45% (IQR: 5%) after 12 months. The PCRpfPR ratios were 0.67 (95% CI: 0.38, 0.91) and 0.74 (95% CI: 0.53, 0.90), respectively. We confirmed the superiority of PBO-LLINs.

Automated phenotyping of mosquito larvae enables high-throughput screening for novel larvicides and offers potential for smartphone-based detection of larval insecticide resistance.

Pyrethroid-impregnated nets have contributed significantly to halving the burden of malaria but resistance threatens their future efficacy and the pipeline of new insecticides is short. Here we report that an invertebrate automated phenotyping platform (INVAPP), combined with the algorithm Paragon, provides a robust system for measuring larval motility in Anopheles gambiae (and An. coluzzi) as well as Aedes aegypti with the capacity for high-throughput screening for new larvicides. By this means, we reliably quantified both time- and concentration-dependent actions of chemical insecticides faster than using the WHO standard larval assay. We illustrate the effectiveness of the system using an established larvicide (temephos) and demonstrate its capacity for library-scale chemical screening using the Medicines for Malaria Venture (MMV) Pathogen Box library. As a proof-of-principle, this library screen identified a compound, subsequently confirmed to be tolfenpyrad, as an effective larvicide. We have also used the INVAPP / Paragon system to compare responses in larvae derived from WHO classified deltamethrin resistant and sensitive mosquitoes. We show how this approach to monitoring larval response to insecticides can be adapted for use with a smartphone camera application and therefore has potential for further development as a simple portable field-assay with associated real-time, geo-located information to identify hotspots.

A dual-target molecular mechanism of pyrethrum repellency against mosquitoes.

Pyrethrum extracts from flower heads of Chrysanthemum spp. have been used worldwide in insecticides and repellents. While the molecular mechanisms of its insecticidal action are known, the molecular basis of pyrethrum repellency remains a mystery. In this study, we find that the principal components of pyrethrum, pyrethrins, and a minor component, (E)-ß-farnesene (EBF), each activate a specific type of olfactory receptor neurons in Aedes aegypti mosquitoes. We identify Ae. aegypti odorant receptor 31 (AaOr31) as a cognate Or for EBF and find that Or31-mediated repellency is significantly synergized by pyrethrin-induced activation of voltage-gated sodium channels. Thus, pyrethrum exerts spatial repellency through a novel, dual-target mechanism. Elucidation of this two-target mechanism may have potential implications in the design and development of a new generation of synthetic repellents against major mosquito vectors of infectious diseases.

Antifungal and Larvicidal Activities of Phlorotannins from Brown Seaweeds.

Phlorotannins are secondary metabolites produced by brown seaweeds with antiviral, antibacterial, antifungal, and larvicidal activities. Phlorotannins' structures are formed by dibenzodioxin, ether and phenyl, ether, or phenyl linkages. The polymerization of phlorotannins is used to classify and characterize. The structural diversity of phlorotannins grows as polymerization increases. They have been characterized extensively with respect to chemical properties and functionality. However, review papers of the biological activities of phlorotannins have focused on their antibacterial and antiviral effects, and reviews of their broad antifungal and larvicidal effects are lacking. Accordingly, evidence for the effectiveness of phlorotannins as antifungal and larvicidal agents is discussed in this review. Online databases (ScienceDirect, PubMed, MEDLINE, and Web of Science) were used to identify relevant articles. In total, 11 articles were retrieved after duplicates were removed and exclusion criteria were applied. Phlorotannins from brown seaweeds show antifungal activity against dermal and plant fungi, and larvicidal activity against mosquitos and marine invertebrate larvae. However, further studies of the biological activity of phlorotannins against fungal and parasitic infections in aquaculture fish, livestock, and companion animals are needed for systematic analyses of their effectiveness. The research described in this review emphasizes the potential applications of phlorotannins as pharmaceutical, functional food, pesticide, and antifouling agents.